WO2009001975A1

WO2009001975A1 - Novel hepatitis c virus mutants having enhanced replication and infectivity

Info

Publication number: WO2009001975A1
Application number: PCT/KR2007/003123
Authority: WO
Inventors: Byung-Yoon Ahn; Ju-Il Kang
Original assignee: Korea University Industry and Academy Cooperation Foundation
Priority date: 2007-06-27
Filing date: 2007-06-27
Publication date: 2008-12-31

Abstract

Disclosed are variants of HCV genotype 2a JFH-I, with enhanced replication and infectivity. Each of the mutants has an amino acid substituted for an amino acid residue located in the C-terminal region of NS5A or in the N-terminal region of NS5B of JFH-1. Compared to JFH-1, the mutant shows alleviated cytotoxicity, but is significantly improved in replication and infectivity. The virus particles produced by the mutant are also highly infectious to hepatic cells and thus can be useful in the development of therapeutic medicines for HCV-induced diseases.

Description

[DESCRIPTION]

[invention Title]

NOVEL HEPATITIS C VIRUS MUTANTS HAVING ENHANCED REPLICATION AND INFECTIVITY

[Technical Field]

The present invention relates to novel hepatic virus (HCV) with enhanced replication efficiency and infectivity.

[Background Art]

Hepatitis C virus (HCV) is a positive (+) sense single strand RNA virus in the family Flaviviridae, and is a pathogen causing acute and chronic hepatitis. Hepatitis C virus (HCV) infects over 170 million people worldwide. Chronic infection occurs in about 50% of cases. It is reported that 20% of chronically infected patients develop cirrhosis over a period of 10 ~ 20 years and some of them are at risk of eventually developing hepatocellular carcinoma (Lauer, G. M. and B. D. Walker (2001) . "Hepatitis C virus infection." N. Engl. J. Med 345(1) : 41-52) .

Based on genetic differences between HCV isolates, hepatitis C virus species are classified into many types, with several subtypes within each genotype or serotype. According to the phylogenetic analysis suggested on the basis of the nucleotide sequence divergence by Simmonds et al . , for example, HCV variants are classified into six genotypes, numbered from 1 to 6, which are further divided into many subtypes, denoted by alphabetic characters (e.g., subtype; Ia, Ib, Ic, 2a, 2b, 2c, 2k, 3a, 3b, 3k, 4a, 5a, βa, 6b, 6d, 6g, 6h, 6k) (Simmonds, P. et al . , (2005) . "Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes." Hepatology 42: 962-973) . Entire genomic nucleotide sequences of most HCV genotypes are revealed

(Choo et al., Science, (1989) 244, pp. 359-362; Kato et al.,

J. Med. Virol., (1992), pp. 334-339; Okamoto, H. et al., J. Gen.

Virol., (1992) 73, pp. 673-679; Yoshioka et al., Hepatology, (1992) 16, pp. 293-299) . The HCV RNA genome is a positive (+ ) sense single strand about 9.6 kb long in length and has a single long open reading frame encoding a polyprotein of -3,010 amino acids, which is posttranslationally cleaved into at least 10 polypeptides (core, El, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins) by both viral and cellular proteases (Lindenbach, B. D. and C. M. Rice (2005) . "Unraveling hepatitis C virus replication from genome to function. " Nature 436(7053): 933-8).

The currently prevalent treatment of hepatitis C is monotherapy with interferon-α or interferon-β or combination therapy with interferon-α and ribavirin, a purine nucleoside derivative. However, therapeutic effects are detected only in about 60% of patients treated with those therapies. Further, after treatment cessation, the disease was found to recur in more than half of the patients who had improved. The efficacy of interferon is known to depend on HCV genotypes, and the attainable therapeutic effects are somewhat high for genotype 2a and low for genotype Ib (Mori, S. et al., Biochem. Biophys. Res. Commun., (1992) 183, pp. 334-342).

Taking into consideration the fact that hepatitis C shows high morbidity in developed countries and eventually leads to death, but is not controlled by commonly usable therapy, it is very important to develop effective therapeutic or preventive drugs for hepatitis C. Therefore, there is a desperate need for an advance in the development of chemical therapy or vaccines specific for HCV or anti-HCV medicines. In this regard, the suppression of HCV replication or the suppression of cell infection with HCV is considered a target for developing anti-HCV medicines .

Since the discovery of HCV in 1989, there have been difficulties in the study of HCV replication mechanisms and HCV infection mechanisms, not only because HCV is difficult to induce to proliferate in cell culture systems and to infect cultured cells, but also because animal tests have been limited to chimpanzees only. Recently, it was reported that a subgenomic replicon consisting of some viral gene responsible for HCV genotype Ib replication and a selectionmarker is able to replicate itself in the human hepatoma cell line Huh-7 (Lohmann, V. , F. Korner, et al. (1999) . "Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line." Science 285(5424) : 110-3) . However, this synthetic subgenomic replicon system has the problem of being unable to produce viral particles because it does not encode structural proteins essential for the production of the virion. Hence, investigation with the synthetic subgenomic replicon system is limited only to the evaluation of intracellular viral RNA replication, but cannot extend to the intracellular production and extracellular release of HCV particles nor to infectivity for new host cells.

Since then, many techniques for using the entire genomic RNA of HCV, but not subgenomic replicons, in the production of virions in cell culture systems have been reported (Lim SP. et al., Virology, 303(2002) pp. 79-99; WO 05080575A1). Such techniques, however, are low in the production yield of virus particles, and nowhere is the infectivity of the produced virus particles reported in detail . Furthermore, it has been reported more recently that Huh-7 cells transfeeted with the entire genomic RNA of JFH-I, a genotype 2a strain, can produce viral particles at high yield, which can be propagated effectively in the cells and can also establish an infection (Wakita et al., Production of infectious hepatitis C virus in tissue culture from a cloned viral genome, Nature Medicine, vol. 11, Number 7, pp. 791-796, July 2005). Moreover, the production of virus particles by other genotype' s RNA or intergenotypic chimera' s RNA was also reported (Cai, Z., C. Zhang, etal. (2005) "Robust production of infectious hepatitis C virus (HCV) from stably HCV cDNA-transfected human hepatoma cells. " J Virol 79 (22) : 13963-73; Heller, T. , S. Saito, etal. (2005) "An in vitro model of hepatitis C virion production . " Proc. Natl. Acad. Sci. U. S. A. 102(7): 2579-83; Lindenbach, B. D. , M. J. Evans, etal. (2005) "Complete replication of hepatitis C virus in cell culture." Science 309(5734): 623-65; Wakita, T., T. Pietschmann, et al. (2005) "Production of infectious hepatitis C virus in tissue culture from a cloned viral genome . " Nat Med 11 (7) : 791-6; Zhong, J., P. Gastaminza, et al. (2005). "Robust hepatitis C virus infection in vitro." Proc Natl Acad Sci U S A 102 (26) : 9294-9; Pietschmann, T. , A. Kaul, et al. (2006) . "Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras." Proc Natl Acad Sci U S A.).

As such, experimental systems by which examination can be made of the intracellular production and extracellular secretion of HCV particles and the infectivity of the virions on new host cells can be established. However, there still remains a demand for highly efficient HCV particle production systems which are useful in the development and study of anti-HCV medicines. On the other hand, when the virus particles is produced from a cell culture system, but not at a high titer, they were found to be infectious to chimpanzees and rats into which human liver had been transplanted. Zhong et al. showed that an improvement in virus titer can be achieved through the repetitive passage of cells to bring about the persistent replication of JFH-I RNA (Zhong, J., P. Gastaminza, et al. (2006). "Persistent hepatitis C virus infection in vitro: coevolution of virus and host." J. Virol 80(22): 11082-93). In this regard, they established a robust highly efficient in vitro infection system based on a specific Huh-7-derived cell line (Huh-7.5.1), which is more permissive of replication. In such a cellular matrix were detected some cell-culture-adaptive JFH-I variants carrying mutations for one amino acid residue within E2 gene (Zhong, J., P. Gastaminza, et al. (2006) . "Persistent hepatitis C virus infection in vitro: coevolution of virus and host." J.Virol 80(22): 11082-93), mutations for intergenotypic JFH-I chimera with the structural gene of genotype Ia (Yi, M., Y. Ma, et al. (2007) . "Compensatory mutations in El, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus. " J Virol 81 (2) : 629-38), and mutations within genotypes Ib or other types (Abe, K., M. Ikeda, et al. (2007). "Cell culture-adaptive NS3 mutations required for the robust replication of genome-length hepatitis C virus RNA." Virus Res 125(1) : 88-97) .

[Disclosure] [Technical Problem]

Based on the understanding that both viral and cellular factors makes dominion over viral replication yield, intensive and thorough research on the development of anti-HCV medicines, conducted by the present inventors, resulted in the finding that, during passages of Huh-7 cells transfected with the genomic RNA of JFH-I, a genotype 2a strain, cell culture-adaptive mutants are generated with the very frequent occurrence of mutations in the C-terminal region of the NS5A gene and in the N-terminal region of the NS5B gene of the genomic RNA of JFH-I maintained within Huh-7 cells, and that not only did some of the mutants produce virus particles infectious to new cells at far higher yield in a cell culture system than did the parent strain JFH-I, but also the virus particles showed lower hepatic lesions than did the parent strain and thus find useful applications in the study and development of anti-HCV medicines. [Technical Solution]

It is therefore an object of the present invention to provide a novel mutant of JFH-I, an HCV genotype 2a strain, with improved replication and infectivity. The novel mutant has a mutation corresponding to one amino acid substitution in the C-terminal region of NS5A or the N-terminal region of NS5B.

It is another object of the present invention to provide a method for preparing the mutant of the present invention which shows enhanced replication efficiency and infectivity in a cell culture system.

[Description of Drawings]

FIG. 1 shows the infection kinetics of JFH-I HCV during long-term passage on Huh-7 cells which have been subcultured at a passage interval of 3 - 6 days for 5 months after electroporation with 20 μg of JFH-I HCV RNA, and stained through an immonofluorescence assay for NS5A (green) and DAPI (blue) , in a graph (A) where percentages of NS5A positive cell counts, calculated using IPLab v3.6.5 image analysis software (Scanalytics Inc.), are plotted against time (days post-electroporation) and in fluorescence microphotographs (B) of the cells at the times indicated by arrows of FIG. IA.

FIG. 2 shows cell culture-adaptive mutants, as revealed by the base sequencing analysis of PCR products obtained through RT-PCR amplification with the viral RNA extracted from Huh-7 cells inoculated with culture supernatants harvested on day 35 after electroporation, and particularly shows positions and frequency of specific mutations different in base from JFH-I, as revealed by the base sequencing analysis of 22 independent clones produced by RT-PCR amplification and cloning of the C-terminal region of NS5A and the N-terminal region of NS5B. In this figure, the amino acid residues are numbered as in the polyprotein of HCV.

FIG. 3 shows the replication properties of mutant viruses in fluorescence microphotographs (A) taken of Huh-7 cells immunostained for NS5A (green) and DAPI (blue) after Huh-7 cells electroporated with mutant HCV RNA were subcultured at a split ratio of 1:8 at passage intervals of 3 days, in a line graph (B) plotted for percentages of NS5A-positive cell counts as a result of flow cytometry against days post-electroporation, in a bar graph (C) plotted for viral infectivity titers in the supernatant of a cell culture on day 4 post-electroporation as a result of a TCID₅₀ assay for various mutants, and in a photograph (D) taken of Northern blots of mutant viral RNAs in cells on day 1, 3 and 5 post-electroporation.

FIG. 4 shows the infectivity of progeny virus particles after the inoculation of progeny virus particles released to culture supernatants of electroporated cells at the same dose

(15,000 TCID₅₀) into Huh-7 cells (~5xlO⁵ cells) in fluorescence microphotographs (A) taken of the infected cells which were diluted at ratios of 1:4, 1:32 and l:25624hrsafter the infection, cultured for 3, 6 and 9 days post-infection, and immunostained against NS5A (green) , and in a graph (B) plotted for percentages of NS5A-positive cell counts as a result of flow cytometry against days post-infection. JFH-I (D35) indicates a culture supernatant containing the virus mutants mentioned in FIG. 2.

FIG. 5 shows the RNA polymerase activity of the recombinant NS5B protein in a photograph (A) of a 10% polyacrylamide gel on which the recombinant NS5B proteins were electrophoresed and stained with Coomassie Brilliant Blue G-250, and in an autoradiograph (B) of a formaldehyde agarose gel on which RNAs newly synthesized with radioisotopes in the presence of the recombinant NS5B proteins with HCV RNA serving as a template were electrophoresed, along with a marker on the left side.

FIG. 6 shows hepatic lesions caused by HCV in a photograph

(A) taken of Huh-7 cells on day 4 after electroporation with each mutant RNA and in a bar graph (B) plotted for growth degrees of the infected cells, expressed as fold-expansion, a multiple of initial cell count.

FIG. 7 shows expression levels and intracellular positions of NS5A protein in fluorescence microphotographs (A) taken of

Huh-7 cells immunostained against NS5A antibody on day 3 post-electroporation and in a graph (B) plotted for fluorescence intensity analyzed by FACS.

FIG. 8 shows the proteolytic digestion of NS5A proteins by proteases as analyzed by immunoblotting against NS5A, NS3, PARP and HSP70 proteins from Huh-7 cell lysates (45 μg protein) on day 3 post-electroporation. Protein positions are indicated by arrows and main cleavage products are marked by asterisks.

[Best Mode]

In accordance with an aspect thereof, the present invention provides a novel mutant of the hepatitis C virus (HCV) genotype 2a JFH-I, with an improvement in replication efficiency and infectivity. Because it is mutated at a position in the 3' -terminal region of the NS5A gene or the 5' -terminal region of the NS5B gene within the genomic RNA of JHF-I, the novel mutant of the present invention has an amino acid substituent for one of the amino acid resides in the polyprotein expressed by the parent strain JFH-I. With the substituted amino acid residue, the novel HCV JFH-I variant can produce virions having significantly improved replication and infectivity.

In greater detail, the novel HCV JHF-I variant in accordance with the present invention is a cell culture-adaptive strain identified during long-term passages of the human hepatoma cell line Huh-7 transfected with the genomic RNA of JFH-I . The variant has improved replication efficiency, for example, increased up to 20-fold in a virion titer, and improved infectivity compared to the parent strain JFH-I.

In a preferred embodiment of the present invention, the mutant of the present invention has a genomic RNA encoding a polyprotein with an amino acid substituent for one of the amino acid residues in the C-terminal region of NS5A or in the N-terminal region of NS5B of the polyprotein encoded by thegenomic RNA of JFH-I. More preferably, the mutant of the present invention has an amino acid substituted for an amino acid located in the N-terminal region of NS5B of the parent strain JFH-I or for an amino acid located in the C-terminal region of NS5A of JFH-I. In a particular preferable embodiment, the novel mutant of the present invention has genomic RNA encoding a polyprotein with an amino acid substituent for one of the amino acid residues at positions from 2340 to 2442 in the polyprotein encoded by the genomic RNA of JFH-I, and preferably with threonine substituted for alanine at position 2343, with arginine substituted for tryptophan at position 2429, with alanine substituted for threonine at position 2431, with glycine substituted for aspartic acid at position 2436, with alanine substituted for threonine at position 2438, with alanine substituted for threonine at position 2439, with alanine or methionine substituted for valine at position 2440, or with arginine, serine or tyrosine substituted for cysteine at position 2441. In another particular preferable embodiment, the novel mutant of the present invention has a genomic RNA encoding a polyprotein with an amino acid substituent for one of amino acid residues at positions from 2460 to 2480 in the polyprotein encoded by the genomic RNA of JFH-I, andpreferablywith valine substituted for leucine at position 2463, with serine substituted for leucine at position 2468, with glycine substituted for serine at position 2469, with glutamine substituted for arginine at position 2474, with histidine substituted for tyrosine at position 2475, or with arginine, proline or leucine substituted for histidine at position 2476. Particularly preferable is a mutant with serine substituted for lysine at position 2468 (L2468S), or with leucine substituted for histidine at position 2476 (H2476L) , the former (L2468S) being more preferable.

The HCV variants according to the present invention can be readily produced using gene engineering techniques known in the art, including the mutation of nucleotides coding for target amino acid residues to nucleotides coding for amino acid residues of interest in the genomic RNA of HCV JFH-I. In greater detail, codons encoding target amino acid residues are substituted with those encoding amino acid residues of interest in the genomic RNA of HCV JFH-I using gene manipulation techniques, followed by the transfection of the substitution-mutated genomic RNA of HCV JFH-I into host cells. The human hepatoma cell line Huh-7 is preferable as the host cell, but the present invention is not limited thereto.

Techniques for the codon substitution, the introduction of the substituted genomic RNA of HCV JFH-I into host cells, and cell culture and culture conditions may be adopted from those known in the art . Thus, in accordance with another aspect thereof, the present invention provides a method for preparing the virus variant of the present invention, which has enhanced replication in and infectivity for cells sufficient for use in the development and study of anti-HCV therapeutics. It is known that the yield of HCV replication is regulated by both cellular and viral factors. It was reported in previous studies with HCV subgenomic replicons that the Huh-7 cell lines in different passages were found to have up to 100-fold differences in their ability to support replicon amplification (Lohmann, V., S. Hoffmann, etal. (2003). "Viral and cellular determinants of hepatitis C virus RNA replication in cell culture." J. Virol 77(5): 3007-19). In this regard, the analysis of the genomic or subgenomic replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that substantially enhance RNA replication and infectivity (Bartenschlager, R., M. Frese, et al. (2004). "Novel insights into hepatitis C virus replication and persistence." Adv Virus Res 63: 71-180; Yi, M., Y. Ma, et al. (2007). "Compensatory mutations in El, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus." J. Virol 81(2) : 629-38) . During passages of a Huh-7 cell line transfected with the genomic RNA of HCV JFH-I, the present inventors identified novel cell-culture-adaptive variants, previously unknown, which surprisingly have mutations collectively generated in the C-terminal region of NS5A or the N-terminal region of NS5B in the genomic RNA of JFH-I. The collective mutation in the C-terminal region of NS5A or in the N-terminal region of NS5B had not been expected at all, and no light has been shed on the reason for the collective mutation. Nonetheless, the cell culture-adaptive variants of the present invention, especially L2468S and H2476L are, in our knowledge, important bases on which a better understanding of the enhancement of replication efficiency duringpassages can be attained. These two variants, which have mutations at the N-terminal region of NS5B, show persistent infectivity over a significantly long period of time, with conspicuously higher virion titers and lower cytotoxicity than the parent JFH-I or the other variants.

The influence of cell culture-adaptive mutations on viral infectivity still remains unknown. Over a long period of time

(59 days or more after electroporation) , the G45IRmutant viruses, which have a mutation in the E2 gene of the JFH-I possessed by

Huh-7.5.1, were found to have higher infectivity and lower dependence on the cell surface CD81 molecules in the blockage of CD81 antibody against viral infection than JFH-I (Zhong, J.,

P. Gastaminza, et al. (2006). "Persistent hepatitis C virus infection in vitro: coevolution of virus and host." J. Virol.

80 (22) : 11082-93) . In contrast to the variants according to the present invention, however, G451R shows a furthermore accelerated cytopathic effect. A difference in viral infectivity was observed between intergenotypic JFH-I chimeras having structural genes from different genotypes (Pietschmann, T., A. Kaul, et al. (2006). "Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras." Proc Natl Acad Sci U. S. A.) . Although chimeras were similar level of core proteins and intracelluar RNA in the early stage after electroporation, significant differences in the release of core proteins were observed therebetween, indicating different activities of the structural proteins in viral assembly and release. The present inventors expected the variants of the present invention to show higher virus RNA replication rates than the wild-type JFH-I. In vitro results, however, suggested that the expectation thereof was not reasonable even if this can be otherwise in vivo. Instead, the present inventors interpreted the result to mean that the alleviation of cytotoxic or cytostatic effects contributes to a significant acceleration in viral expansion and infection. In addition, with reference to FIG.4, an increase in viral infectivity is based on an increase in the specific infectivity of secreted virus particles, rather than on an increase in the number of secreted virus particles.

Viral infection and replication is disadvantageous for cell growth. Highly efficient replication causes the accumulation of viral components that have not yet been assembled, thus interrupting the assembly or extracellular release of virus particles. It may also cause ER (endoplasmic reticulum) stress or even induce the apoptosis of the infected cells . In this regard, mutants which impose less burden on cells or are less disadvantageous to cell growth are allowed to efficiently increase in number in culture systems . Without regard to concrete mechanism, the L2468S mutant virus according to the present invention is believed to impose less burden on cells than does JFH-I. As for the H2476L mutant, the first discovery thereof in subgenomic JFH-I has been made known, and the colony formation efficiency was increased three-fold with H2476L mutated replicon compared with the original JFH-I (Kato, T. , T. Date, et al . (2003) . "Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon." Gastroenterology 125(6): 1808-17). A further study on whether JFH-I infection causes a cytotoxic effect or gives a disadvantage to cell growth is needed.

The caspase-mediated cleavage of the NS5A protein was reported (Goh, P. Y., Y. J. Tan, et al. (2001). "The hepatitis C virus core protein interacts with NS5A and activates its caspase-mediated proteolytic cleavage." Virology 290(2): 224-36; Kalamvoki, M. and P. Mavromara (2004) . "Calcium-dependent calpain proteases are implicated in processing of the hepatitis C virus NS5A protein. " J Virol 78 (21) : 11865-78; Kalamvoki, M., U. Georgopoulous, etal. (2006) . "The NSδAprotein of the hepatitis C virus genotype Ia is cleaved by caspases to produce C-terminal-truncated forms of the protein that reside mainly in the cytosol." J. Biol. Chem. 281(19) : 13449-62) . However, nowhere was evidence of HCV replication-induced apoptosis found in these studies. As understood from the caspase-mediated activation or inhibition of many viral proteins (Best, S. M. and M. E. Bloom (2004). "Caspase activation during virus infection: more than just the kiss of death?" Virology 320(2) : 191-4), when account is taken of various functions of the NS5A protein, the cleavage of the NS5A protein is thought to be a normal process associated with the regulation of NS5A activity. However, the alleviated NS5A cleavage observed in the mutants having alleviated cytotoxicity according to the present invention suggested that the cleavage of NS5Amay result from cytotoxicity.

In the following examples, it is confirmed that NS5B plays an important role in the regulation of viral infection. The mechanism by which one amino acid substitution in NS5B alleviates cytotoxicity and thus enhances infectivity still remains unclear. According to the crystal structure of NS5B (Lesburg, C. A., M. B. Cable, et al. (1999) , "Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site." Nat Struct Biol 6(10) : 937-43), the two mutations L2468S and H2476L in accordance with the present invention, as in many cell culture-adaptive mutants found in subgenomic replicons of other genotypes, seem to be located at the surface of molecules distant from the active site of the enzyme . Mutations reported to increase about 1, 000-fold in the colony formation efficiency of subgenomic replicons, such as R884G, fall within this range (Lohmann, V., F. Korner, et al. (2001). "Mutations in hepatitis C virus RNAs conferring cell culture adaptation." J Virol 75(3): 1437-49; Bartenschlager, R., M. Frese, et al.

(2004) . "Novel insights into hepatitis C virus replication and persistence." Adv Virus Res 63: 71-180) . Both L2468 and H2476 are conserved in all HCV genotypes, and no substitutions therefor were found in vivo. It can be inferred that cell culture-adaptive mutation takes place not only outside the active site of the enzyme due to the indispensability of the enzymatic function for RNA replication, but also near the potential position at which interactions with other proteins occur.

Viral infectivity is also believed to be regulatedby complex interactions between viral proteins as well as by the properties of each viral protein. NS5A is a possible one of the proteins that closely interact with NS5B (Shirota, Y., H. Luo, et al. (2002) . "Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B andmodulates RNA-dependent RNApolymerase activity." J. Biol. Chem 277(13): 11149-55). The interaction between NS5A and NS5B is reported to be responsible for virus replication (Shimakami, T., M. Hijikata, etal. (2004). "Effect of interaction between hepatitis C virus NS5AandNS5B on hepatitis C virus RNA replication with the hepatitis C virus replicon." J. Virol.78(6): 2738-48) . The present invention, featuring the collective mutations at the C-terminal region of NS5A or the N-terminal region of NS5B, is coincident with these reports. On the other hand, the mutations occurring within NS5A are quite different from those found predominantly at the hyperphosphorylation region near ISDR in the subgenomic replication (Blight, K. J., A. A. Kolykhalov, et al. (2000). "Efficient initiation of HCV RNA replication in cell culture." Science 290(5498) : 1972-4).

[Mode for Invention]

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

1. Materials and Methods

(1) Cell culture Human hepatoma Huh-7 cell lines were cultured in Dulbecco' s Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 25 mM HEPES, 100 U/ml penicillin G, 100 μg/ml streptomycin and 5% fetal bovine serum.

(2) Plasmid construction The pJFHl plasmid, which carries the entire genomic RNA of JFH-I, a strain of HCV genotype 2a, was provided by Dr. T. Wakita (Takaj i Wakita et al . , Production of infectious hepatitis C virus in tissue culture from a cloned viral genome, Nature Medicine, Vol. 11, Number 7, July 2005) .

In order to introduce the mutations identified in cell culture-adaptive viruses in accordance with the present invention, a SanDI-RsrII fragment or an RsrII-Hpalp fragment of the sequence amplified from the mutant viruses was substituted for the corresponding nucleotide sequence of the pJFH plasmid. The H2476L mutation identified in the subgenomic replicon pSGR-JFHl-H2476L (Kato, T., T. Date, et al. (2003). "Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. " Gastroenterology 125 (6) : 1808-17) was introduced into pJFHl by substitution with Spel-Xbal fragments. As for the replication-deficient GND mutation ( (Asp²⁷⁶⁰→Asn) of NS5B (Yamashita, T., S. Kaneko, et al. (1998). "RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus

NS5B protein truncated at the C-terminal region. " J. Biol. Chem.

273(25): 15479-86), it was prepared using two-step PCR and substituted at the corresponding HindIII cleaved site of pJFH-1.

(3) HCV RNA Synthesis and Electroporation The electroporation of viral RNAwas conducted using a method known in the art (Lindenbach, B. D., M. J. Evans, et al. (2005) . "Complete replication of hepatitis C virus in cell culture." Science 309(5734): 623-6; Wakita, T., T. Pietschmann, et al. (2005) . "Production of infectious hepatitis C virus in tissue culture from a cloned viral genome." Nat. Med. 11(7): 791-6; Zhong, J., P. Gastaminza, et al. (2005). "Robust hepatitis C virus infection in vitro. " Proc. Natl. Acad. Sci. U. S. A.102 (26) : 9294-9) . In brief, pJFHl was cleaved with Xbal and treated with mung bean nuclease to produce a precise 3' end of virus RNA. The linear DNA thus formed was used as a template for in vitro transcription with MEGAscript™ T7 kit (Ambion) to synthesize HCV RNA. After separation into single cells by trypsinization, Huh-7 cells were resuspended at a density of 1 x 10⁷ cells/ml in a HEPES-buffered medium (2 mM Hepes, 15 mM potassium phosphate buffer, 250 mM mannitol, 1 mM MgC12, pH 7.2) (Zheng, Q. A. and D. C. Chang (1991) . "High-efficiency gene transfection by in situ electroporation of cultured cells." Biochim Biophys Acta 1088(1) : 104-10) . Electroporation was carried out on a 400-μl volume of the cell suspension mixed with 20 μg of the HCV RNA using 5 bursts of 350 V, 100% modulation, 40 kHz RF, with 5 ms duration and 1 s burst interval in a Gene Pulser^R II RF module (Bio-Rad) .

(4) Sequence analysis of viral RNA

Viral RNA was prepared from HCV-infected cells or cell cultures using a QIAamp^R viral RNA mini kit (Qiagen) and converted into cDNA using Superscript™ II reverse-transcriptase ( Invitrogen) .

20 fragments that covered the entire genome of HCV were amplified by PCR, immediately followed by base sequencing using ABI 3100 genetic analyzer (Applied Biosystems). The 5'- and 3' -end of the sequence was determined by 5'- and 3'-RACE (Kato, T., A. Furusaka, et al. (2001). "Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient." J. Med. Virol. 64(3) : 334-9). Some PCR products which were difficult to analyze for base sequence due to the reiteration of the same bases were cloned into a pMD18-T vector (Takara) before sequencing.

(5) Antibodies and Immunoblotting Anti-NS5A and anti-NS3 polyclonal antibodies were obtained by immunizing rabbits with the recombinant proteins expressed in E.coli.

Anti-Hsp70 (W-27) antibodies and anti-PARP (H-250) antibodies were purchased from Santa Cruz Biotechnologies Inc.

As anti-rabbit IgG (NA934V) and anti-mouse IgG (NA931V) HRP

(horseradish peroxidase) -conjugated antibodies, products from

GE Healthcare were used.

For immunoblotting, cells were disrupted in a triple detergent lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl,

1 mM EDTA, 0.5% Triton X-IOO, 0.1% SDS, 0.25% deoxycholate, 1 mM phenylmethylsulphonyl fluoride) .

After separation on denaturing polyacrylamide gel, proteins were transferred onto a nitrocellulose membrane (Hybond ECL; Amersham Biosciences) .

The proteins were sequentially reacted with dilutions of a primary and a secondary antibody in a TBST buffer ( 1OmM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) containing 5% skim milk, followed by the visualization of blots with an enhanced chemiluminescent reagent (PicoWest SuperSignal ECL Substrate; Pierce) and exposure to an X-ray film.

(6) Immunofluorescence and Virus Titration Electroporated, that is, infected Huh-7 cells were grown on a cover slip and fixed by treatment for 10 min with a PHEM buffer (6OmMPIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgSO₄, pH 7.0) containing 3.7% paraformaldehyde. After the cells were treated for 10 min with PBS containing 0.5% Triton X-100 to increase membrane permeability, they were reacted for 10 min with blocking buffer (PBS containing 1% BSA and 0.1% Tween-20) . Subsequently, the cells were incubated at 37⁰C for 2 hrs with an anti-NS5A rabbit polyclonal antibody (dilution 1:1000) in the blocking buffer. After washing with PBS, the cells were incubated at 37⁰C for 1 hr with a 1:1000 dilution FITC-conjugated goat anti-rabbit IgG (F1262, Sigma-Aldrich) in the blocking buffer. Nuclear DNA was visualized by DAPI staining (PBS containing 1 μg/ml of DAPI) forlmin. The cover slip was washed with PBS, dried, and mounted on a slide glass using FluoroGuard™ antifade reagent (Bio-Rad) before observation under a fluorescence microscope (Axioscope 2 plus; Carl Zeiss) .

The titer of infectious viruses in a cell culture was determined by a TCID₅₀ assay. Huh-7 cells grown to 70 ~ 80% confluency in 96-well plates were inoculated with 50 μl of a serial 5-fold dilution of virus stock (each dilution was performed in sextuple wells) . After viral attachment at 37⁰C for 1 hr, 100 μl of fresh medium was added to each well and the cells were further incubated for 3 days. Cells were fixed with absolute methanol at room temperature for 20 min and immunostained before the observation of the positive cells with an inverted fluorescence microscope (Carl Zeiss) . TCID₅₀ titers were calculated using the Reed-Muench technique. (7) Flow Cytometry

The trypsinized cells were resuspended at a density of 2^χlO⁶ cells/ml, fixed with PBS containing 2% paraformaldehyde at 4⁰C for 1 hr, and then treated with PBS containing 0.2% Tween-20 at normal temperature for 15min to increase membrane permeability. Afterwards, the cells were reacted at 37 °C for 2 hrs with a 1:500 dilution of an anti-NS5A antibody in a staining buffer (PBS containing 1% BSA and 0.1% Tween-20) . After washing with PBS containing 0.1% Tween-20, the cells were incubated at 37⁰C for 1 hr with a 1 : 500 dilution of an FITC-conjugated goat anti-rabbit IgG (F1262; Sigma-Aldrich) and were counted using an FACSCalibur flow cytometer (BD Bioscience) .

(8) Northern blotting

20 μg of the total RNA from the Huh-7 cells electroporated with HCV RNA was separated on a 1% formaldehyde agarose gel and transferred to a positive nylon membrane (Nytran supercharge;

Schleicher & Schuell) , followedby fixation using a UV crosslinker at 20,000 μJ/cm². [α-³²P]dCTP probes for HCV RNA detection were synthesized using a random prime DNA labeling kit (Takara) with an NS5A gene region serving as a template. The membrane was incubated with a hybridization solution (7% SDS, 1 mM EDTA, 0.5 M sodium phosphate, pH 7.2) containing the probes and exposed to an X-ray film.

(9) Expression and Purification of Recombinant NS5B protein The NS5B protein, lacking 21 hydrophobic, C-terminal amino acid residues, was expressed as a protein with six histidines tag in the BL21-CodonPlus (DE3) -RIL, an E. coli strain, transformed with pET-28a (Novagen) . Protein expression was induced by treating the cells with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 25°C for 6 hrs . The recombinant protein was purified by metal affinity chromatography using Ni-NTA agarose resin (Qiagen) , concentrated in a spin concentrator (30 K MWCO; Vivascience) , and stored at -7O⁰C in a storage buffer (10 mM Tris-HCl, pH 7.5, 5 mM dithiothreitol, 600 mM NaCl, 40% glycerol) .

(10) In vitro Activity of RNA replicase (RdRp) The in vitro activity of RNA replicase was evaluated according to a method well known in the art (Lohmann, V. , F. Korner, et al. (1997). "Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity." J. Virol 71 (11) : 8416-28; Oh, J. W., T. Ito, et al. (1999). "A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA." J Virol 73(9) : 7694-702) . In brief, 0.5 μg of the purified NS5B-ΔC21 protein was incubated with 500 ng of full-length JFHl RNA in 50 μl of a reaction buffer (20 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM dithiothreitol, 25 mM KCl, 1 mM EDTA, 20 U RNasin (Promega), 25 ug/ml actinomycin-D, 5 μCi [α-³²P]UTP (3000 Ci/mmol; Perkin Elmer) , 50 μMcoldUTP and 500 μM each NTP) . Reaction was performed at 22⁰C for the indicated time period, and was terminated by digestion at 37°C for 30 min with protease K (1 mg/ml protease K, 50 mM Tris-HCl pH 7.5, 15OmMNaCl, 0.5% SDS, 0.1 mg/ml yeast tRNA) . The reaction mixture was extracted with the same volume of acidic phenol/chloroform, followed by RNA precipitation in isopropanol and RNA separation on 1% formaldehyde/agarose gel. The gel was dried on a nylon membrane (Schleicher & Schuell) and exposed to an X-ray film. 2 . Results

(1) HCV Infection Kinetics during Long-term Passage Huh-7 cells electroporated with in vitro-transcribed JFHl RNA were subcultured for 5 months with passage intervals of 3 ~ 6 days. Cells with an HCV replicon were monitored by immunofluorescence using an anti-NS5A antibody for each passage and the results are shown in FIG. 1. As seen in FIG. 1, the percentage of NS5A positive cells was evaluated to be 35% or less of the electroporated cells on day 2 post-electroporation, but greatly decreased to around 5% on day 10 post-electroporation, and then sharply increased to 90% or greater over 20 days. In this first increase period, some cells floated as round single cells. After that time, the percentage of NS5A positive cells dropped to about 50%, after which a rise to nearly 100% occurred. The viral infection was generally maintained at the level of no or slight hepatic lesions. In another assay, a first peak was observed, after which the percentage did not peak, but continuously decreased (data not shown) .

The biphasic infection kinetics observed by the present inventors was similar to that reported for the Huh-7.5.1 cell system (Zhong, J., P. Gastaminza, et al . (2006). "Persistent hepatitis C virus infection in vitro: coevolution of virus and host." J. Virol 80(22) : 11082-93) . However, the frequency and severe fluctuation of infectivity observed in the prior art were not found in the experiments according to the present invention.

(2) Identification of Culture-adaptive mutant In order to examine the infectivity of the virus released into culture media at the first peak (around day 35 post-electroporation) during the long-term passage of the cells electroporated with JFHl RNA, the culture media were inoculated into normal Huh-7 cells and the rate of viral infection throughout all of the cells was measured. As a result, the viral spread rate was found to be higher than that of the virus harvested in the early stage of electroporation (data not shown) .

The higher spread rate of the viruses at this time point was inferred to result from the appearance of mutant viruses having enhanced infectivity. The supernatants of the cell culture media at this time point were inoculated and infected into naive Huh-7 cells so as to amplify only the viruses which were enhanced in infectivity. Total RNA was extracted from the infected cells and HCV RNA was amplified using RT-PCR, followed by the sequence analysis of the PCR product.

The RNA sequences of viruses were conserved at high levels over the entire genome, except for combined bases found at various positions in the C-terminal region of NS5A and the N-terminal region of NS5B. Each mutant was identified by cloning amplified DNA and base-sequencing the clone . From 22 clones, 18 different missense mutations were detected at 14 positions distributed within the regions, among which 11 mutations were located in the C-terminal region of NS5A, with 7 mutations present in the N-terminal region of NS5B (FIG.2). Of the mutations identified, L2468S within NS5B showed the highest frequency (50%) , followed by A2343T (18%) within NS5A and Y2475H (14%) within NS5B.

(3) Infectivity of Culture-Adaptive Mutants The three most frequent mutations, A2343T, L2468S and Y2475H, were introduced into JFHl RNA, and Huh-7 cells were electroporated with each kind of RNA so as to analyze the effects of the mutations . In addition, the same processes for RNA construction and electroporation were applied both to the GND mutant deficient of the replication of NS5B (Yamashita, T., S. Kaneko, et al. (1998) . "RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region." J. Biol. Chem. 273(25): 15479-86) and the adaptive mutant H2476L previously reported in NS5B of the subgenomic replicon.

Viral spread was monitored using an immunofluorescent assay (FIG. 3A) , and cells were quantitatively analyzed by flow cytometry with treatment of cell passages with an anti-NS5A antibody every third day for 12 days (FIG. 3B) . All of the constructs, except for GND, were found to express NS5A in 20

~ 50 % of the cells in the early stage following electroporation. The infection kinetics of JFHl, A2343T, and Y2475H greatly decreased from three days post-electroporation, whereas the spread of the mutants L2468S and H2476L, although somewhat retarded in early stages, was increased to around 80% of the total cell count. On day 4 post- electroporation, the titer of the concentration of virus particles released into the cell culture supernatant was measured to amount to about 20,000 TCID₅o/ml for L2468S, which was about 20 times as much as that of JFHl (FIG. 3C) . The mutants A2343T and Y2475H were similar in viral titer to JFHl, while the viral titer provided by the H2476L was about four times as high as that provided by naive JHF-I. As for intracellular RNA replication, it was drastically accelerated at the early stage in JFHl and the two mutants (A2343T and Y2475H) and sharply decreased from 3 days post-electroporation. In contrast, the mutants H2476L and L2468S were found to steadily increase in replication rate, although this was slower than other viruses. These RNA replication patterns are similar to the increasing pattern of NS5A positive cell counts, as measured by immunofluorescence assay (FIG. 3D) .

For the comparison of infectivity, progeny virus particles of each variant, produced through electroporation, were inoculated at the same dose (15,000 TCID₅₀) into Huh-7 cell (~5 X 10⁵ cells) . In order to minimize the inhibitory effect of cell confluence on virus replication (Nelson, H. B. and H. Tang (2006) . "Effect of cell growth on hepatitis C virus (HCV) replication and a mechanism of cell confluence-based inhibition of HCV RNA and protein expression." J. Virol. 80(3): 1181-90), infected cells were separated at split ratios of 1:4, 1:32 and 1:256 24 hrs post-infection, and were analyzed for viral infectivity through flow cytometry and immunofluorescence assay. For comparison, the cell culture supernatant obtained on day 35 after electroporation with JFH-I was used as a control. Although low infection levels were measured in comparison to previous experiments, similar results to the electroporation experiment were detected in overall infection kinetics and differences in infectivity among mutants (FIG. 4).

Taken together, the data obtained above support the present inventors' inference that the variants L2468S and H2476L are significantly higher in infectivity than are JFH-I and other variants, which makes a contribution, at least partially, to the rapid spread of the cell culture-adaptive mutants, such as L2468S, as shown in the electroporation experiment (over cells on day 35 post-electroporation) . As shown in the infection experiment (FIG. 4), an increase in the specific infectivity of released virus particles is due to the increase in the infectivity of L2468S and H2476L. (4) Enzymatic Activity of NS5B Protein

An examination was made of whether the increased infectivity of L2468S and H2476L was attributed to the enzymatic function of the NS5B protein. For this, JFH-I proteins, and H2476L, L2468S, Y2475H and GND recombinant proteins were expressed in E.coli and purified, as taught previously (Ferrari, E., J. Wright-Minogue, et al. (1999). "Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli." J Virol 73(2): 1649-54) (FIG. 5A). RNA replicase (RdRp) activity was assayed in vitro with a full-length JFH-I RNA template, as described previously (Lohmann, V. , F. Korner, et al. (1997) . "Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity." J. Virol. 71(11) : 8416-28).

The size and intensity of the newly synthesized RNA increased with reaction progress (FIG. 5B) . Interestingly, none of the three variants was found to have higher RNA-dependent RNA polymerase activity than JFH-I, indicating that there was no relationship between the catalystic activity of NS5B and viral infectivity. The RNA synthesis is thought to take a de novo pathway rather than a copy-back primed synthesis pathway, such as that reported by Oh and Ito (1999) , because the RNA products are smaller than the RNA template.

(5) Alleviated Cytotoxicity and Enhanced Infectivity Recent studies reported that HCV, especially JFH-I, can cause hepatic lesions in cell cultures (Zhong, J., P. Gastaminza, et al . (2006) . "Persistent hepatitis C virus infection in vitro: coevolution of virus and host." J Virol 80(22): 11082-93). Accordingly, some degree of cell lesion was also observed for the mutants according to the present invention. To specify this observation, electroporated cells were incubated for 4 days, after which the number of viable cells was counted. Cytotoxicity was expressed as fold-expansion, that is, the ratio of the count of viable cells post-infection to initial cell count. Surprisingly, the mutants H2476L and L2468S showed about three-fold expansion, which is, although lower than that of the replication-incapable GND strain, two times as high as that of JFH-I and the other two variants (FIG. 6) . To get a better understanding of viral cytotoxicity, the expression of viral proteins in electroporated cells was examined using an immunofluorescence assay. Interestingly, noticeable differences were found in NS5A protein expression among the adaptive mutants . Whereas most of the cells electroporated with JFHl, A2343T or Y2475H were observed to have highly fluorescent microparticle structures of NS5A proteins and somtimes aggregate around the nucleus, the cells electroporated with H2476L or L2468S showed weak fluorescence distributed over the cytosol (FIG. 7A). In fact, the mean fluorescence intensities of NS5A detected in cells with L2468S and H2476L were 40 % and 56 %, respectively, of that detected in cells electroporated with JFH-I . In the case of the remaining two mutants, they were similar in mean fluorescence intensity to JFH-I (FIG. 7B) . This inverse proportion between viral infectivity and viral protein level also supports the assumption that the enhanced infectivity results from alleviated cytotoxicity. The NS3 protein also exhibited a similar pattern (data not shown) .

For a further investigation of cytotoxicity, NS5A proteins were visualized by immunoblotting. As taught previously (Goh, P. Y., Y. J. Tan, et al. (2001). "The hepatitis C virus core protein interacts with NS5A and activates its caspase-mediated proteolytic cleavage." Virology 290(2): 224-36; Kalamvoki, M. and P. Mavromara (2004) "Calcium-dependent calpain proteases are implicated in processing of the hepatitis C virus NS5A protein." J. Virol. 78(21): 11865-781; Kalamvoki, M., U. Georgopoulous, et al. (2006) "The NS5A protein of the hepatitis C virus genotype Ia is cleaved by caspases to produce C-terminal-truncated forms of the protein that reside mainly in the cytosol." J. Biol. Chem. 281(19): 13449-62), protein hydrolysates with molecular weights of 50, 39, 30 and 25kDa, cleaved from a 58 kDa protein, were found to react with anti-NS5A antibodies. Interestingly, these truncated proteins are not derived from H2476L, and exist at a very small level in L2468S

(FIG. 8) . It was reported that NS5A is cleaved by the cytosol caspase activated upon apoptotic stimulation (Goh, P. Y., Y.

J. Tan, etal. (2001) . "The hepatitis C virus core protein interacts with NS5A and activates its caspase-mediated proteolytic cleavage." Virology 290(2): 224-36; Kalamvoki, M. and P. Mavromara (2004) "Calcium-dependent calpain proteases are implicated in processing of the hepatitis C virus NS5A protein. " J Virol 78(21) : 11865-781; Kalamvoki, M., U. Georgopoulous, et al. (2006) "The NS5A protein of the hepatitis C virus genotype Ia is cleaved by caspases to produce C-terminal-truncated forms of the protein that reside mainly in the cytosol . " J. Biol. Chem. 281(19): 13449-62). However, the proteolytic cleavage of cellular poly (ADP) -ribose polymerase (PARP) was not detected in the experiments of the present invention. Also, no apoptotic fragments of cellular DNA were found (data not shown) . Hence, no evidence of HCV-induced apoptosis, except for NS5A cleavage, was observed. There were no great differences in the expression of viral NS3 and cellular Hsp70 protein among the mutants.

[industrial Applicability] As described hitherto, cell culture-adaptive HCV mutants identified according to the present invention are improved in replication efficiency and infectivity, compared to naive HCV. Accordingly, the mutants in accordance with the present invention may be useful in the study of mechanisms of the replication and infectivity of HCV, and furthermore, in the development of anti-HCV medicines.

Claims

[CLAIMS]

[Claim l]

A mutant of hepatitis C virus (HCV) genotype 2a JFH-I, which has an amino acid substituent for one of amino acid residues at positions from 2340 to 2442 or at positions from 2460 to 2480 in a polyprotein sequence encoded by a genomic RNA of the hepatitis

C virus genotype 2a JFH-I.

[Claim 2] The mutant according to claim 1, wherein the amino acid substituent is threonine for alanine at position 2343, arginine for tryptophan at position 2429, alanine for threonine at position 2431, glycine for aspartic acid at position 2436, alanine for threonine at position 2438, alanine for threonine at position 2439, alanine or methionine for valine at position 2440, arginine, serine or tyrosine for cysteine at position 2441, valine for leucine at position 2463, serine for leucine at position 2468, glycine for serine at position 2469, glutamine for arginine at position 2474, histidine for tyrosine at position 2475, or arginine, proline or leucine for histidine at position 2476.

[Claim 3]

The mutant according to claim 2, wherein the amino acid substituent is serine for leucine at position 2468 or leucine for histidine at position 2476.

[Claim 4]

The mutant according to claim 1, wherein the amino acid substituent is serine for leucine at position 2468 (L2468S) .

[Claim 5]

The mutant according to claim 1, wherein the amino acid substituent is leucine for histidine at position 2476 (H2476L) .

[Claim 6] A method for preparing a mutant of hepatitis C virus (HCV) comprising:

(a) mutating a genomic RNA of HCV JFH-I to substitute an amino acid residue located in a C-terminal region of NS5A or in an N-terminal region of NS5B with a different amino acid residue; (b) introducing the mutated genomic RNA of HCV JFH-I into a host cell; and (c) culturing the host cell carrying the mutated genomic RNA of HCV JFH-I, whereby the mutant shows high replication efficiency and infectivity.

[Claim 7]

The method according to claim 6, wherein the host cell is the human hepatoma cell line Huh-7.

[Claim 8] The method according to claim 6 or 7, wherein the mutant has a genomic RNA encoding a polyprotein with an amino acid substituent being threonine for alanine at position 2343, arginine for tryptophan at position 2429, alanine for threonine at position 2431, glycine for aspartic acid at position 2436, alanine for threonine at position 2438, alanine for threonine at position 2439, alanine or methionine for valine at position 2440, arginine, serine or tyrosine for cysteine at position 2441, valine for leucine at position 2463, serine for leucine at position 2468, glycine for serine at position 2469, glutamine for arginine at position 2474, histidine for tyrosine at position 2475, or arginine, proline or leucine for histidine at position 2476 in a polyprotein sequence encoded by a genomic RNA of the hepatitis C virus.

[Claim 9]

The method according to claim 8, wherein the amino acid substituent is serine for leucine at position 2468 or leucine for histidine at position 2476.